The goal of this project is to facilitate rapid diagnosis of drug resistant tuberculosis from sputum at the point of care in primary health care facilities using a handheld, easy to use, inexpensive, closed unit fully integrated nucleic acid testing device. The cost of the envisioned system will be 10-100 times lower and the size will be significantly smaller than current PCR-based fully integrated benchtop systems for point of care nucleic acid testing. The project builds upon ongoing work aimed at developing a similar albeit simpler system for detection of Herpes Simplex Virus from swab samples. The assay is based on the isothermal EXponential Amplification Reaction (EXPAR) coupled to visual detection using lateral flow. Simple and rapid sample preparation utilizes a miniaturized bead blender originally development for mechanical lysis of anthrax spores, which in its current version is also capable of nucleic acid purification. The proposed concept involves a handheld, battery operated instrument, to which a disposable plastic cartridge is attached. The cartridge houses chambers, valves, and fluid conduits for sample preparation, amplification and detection, all in a single, closed system. In its final form, the cartridge will contain all necessary reagents on board. The handheld instrument controls the pumping, valving and isothermal heating steps necessary for test execution. The operator simply attaches the cartridge to the instrument, inserts the sample, initiates the test by pushing a button, and after approximately 30 minutes to one hour reads off the result. Such a system will allow health care professionals in a point-of- care setting to diagnose and treat drug resistant tuberculosis in a timely manner, thereby improving patient outcomes and preventing further spread. Aim 1 of this project focuses on establishing a simple, rapid and effective sample preparation method applicable to Mycobacterium tuberculosis in sputum. Under Aim 2, we will establish multiplexed isothermal nucleic acid amplification assays based on EXPAR, coupled with lateral flow based detection. Aim 3 of this project focuses on developing cartridge and instrument prototypes to execute the sample preparation, isothermal amplification, and visual detection methods developed under aims 1 and 2. Performance verification for tasks described under Aims 1-3 will initially involve BSL2 surrogates and DNA extracts of M. tuberculosis, followed by drug susceptible and resistant M. tuberculosis grown in culture and spiked into TB negative sputum, and ultimately clinical sputum samples that have been tested for M. tuberculosis and antimicrobial susceptibility using established methods. This project presents a unique opportunity to bring together multidisciplinary expertise to develop a next generation of assays and analyzers for nucleic acid testing. In the long term, the assays and devices developed herein can be applied to a spectrum of pathogens relevant to biothreat detection and infectious disease diagnosis in resource-limited settings. The goal of this project is to facilitate rapid diagnosis of drug resistant tuberculosis from sputum at the point of care in primary health care facilities using a handheld, easy to use, inexpensive, closed unit fully integrated nucleic acid testing device. The cost of the envisioned system will be 10-100 times lower and the size will be significantly smaller than current PCR-based fully integrated benchtop systems for point of care nucleic acid testing.